Immunogenic minicells and methods of use

ABSTRACT

The disclosed invention relates to immunogenic minicells cells (anucleated) and their use to induce an immune response from a subject.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/133,308, filed Jun. 4, 2008, which is a divisional of U.S.application Ser. No. 10/832,000, now U.S. Pat. No. 7,396,822, filed Apr.26, 2004, which is a continuation-in-part of U.S. application Ser. No.10/154,951, filed May 24, 2002, now abandoned, which claims priority toU.S. Provisional Application No. 60/359,843, filed Feb. 25, 2002 andU.S. Provisional No. 60/293,566, filed May 24, 2001, all of which areexpressly incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The disclosed invention relates to immunogenic minicells and their useto induce an immune response from a subject.

BACKGROUND OF THE INVENTION

A variety of recombinant protein expression systems have been used toproduce immunogenic compositions. Some commonly used expression systemsinclude the rabbit reticulocyte lysate system, E. coli S30 ExtractSystem (both available from PROMEGA) (Zubay, Methods Enz. 65:856, 1980),eukaryotic cell culture expression, and bacterial expression systems.

Bacterial expression systems are generally similar to that of theeukaryotic expression systems in that they both use the host cellenzymes to drive protein expression from recombinant expression vectors.In bacterial expression systems, bacterial cells are transformed withexpression elements from which transcription is driven. The resultingmessenger ribonucleic acid (mRNA) is translated by the host cell, thusyielding a protein of interest.

Bacteria divide very rapidly and are easy to culture; it is relativelyeasy to produce a large number of bacteria in a short time. Moreover,incorporation of expression elements into bacterial cells is efficient.Cultures of transformed cells can be grown to be genetically identical.Thus, all cells in the culture will contain the expression element.

Bacterial expression systems can be used to produce membrane proteinsfor use in immunogenic compositions. Although bacterial expressionsystems can be used to produce antigenic material, there are a varietyof disadvantages to use such a system. For example, the potential forcontamination of the immunogenic product with live, reproducingbacterial cells renders bacterial expression systems undesirable forproducing immunogenic material. Similar drawbacks exist when immunogeniccompositions are prepared using eukaryotic host cells.

Minicells produced by host cells are advantageous over whole-cellprotein expression systems. Using minicells to produce antigeniccompositions greatly reduced the likelihood of contamination with awhole, live cell. Khachatourians (U.S. Pat. No. 4,311,797) exploited anE. coli strain that constitutively produced anucleated minicells andconstitutively expressed the K99 surface antigen. The resulting E. coliderived minicells were prepared as a vaccine. The vaccine induced theproduction of antibodies against growing and infective enteropathogenicK99+ E. coli in cattle and was, thus effective against coniformenteritis. It is important to note that this reference only teaches theuse of E. coli based minicells to express a naturally occurring E. coligene. Accordingly, there is still a need in the art to produceimmunogenic minicells capable of expressing heterologous genes tostimulate an immunogenic response in a subject. Additionally there is aneed to use minicells to carry vectors encoding an antigen of interestthat are capable of being expressed in the cells of the target host, andnot just in the minicell.

SUMMARY OF THE INVENTION

Embodiments of the invention include methods of generating animmunogenic response in a subject comprising, introducing a minicell toa subject, wherein said minicell comprises a plasmid having an openreading frame encoding an antigen of interest, and a eukaryoticexpression sequence operably linked to said open reading frame, suchthat said antigen of interest is expressed in the subject.

In further embodiments, the plasmid can further comprise a prokaryoticexpression sequence operably linked to said open reading frame, suchthat said antigen of interest is expressed in the minicell and in thesubject. In additional embodiments, the antigen of interest expressed inthe minicell is displayed on the surface of the minicell.

In more specific embodiments, the eukaryotic expression sequence cancomprise a Cytomegalovirus (CMV) promoter. In other aspects, the openreading frame is derived from the genome of a pathogen, including avirus or a bacterium, such as Bacillus anthracis, for example. In otheraspects, the open reading frame encodes an antigen characteristic of acancerous cell.

Additional aspects include methods of generating an immunogenic responsein a subject comprising, introducing a minicell to a subject, whereinsaid minicell comprises first and second open reading frames, encodingfirst and second antigens respectively, and a eukaryotic expressionsequence operably linked to said first open reading frame and aprokaryotic expression sequence operably linked to said second openreading frame, such that said first antigen is expressed in the subject,and said second antigen is expressed in the minicell.

In additional aspects, the first and second open reading frames can belocated in a plasmid. The first and second open reading frames can belocated in the same plasmid or different plasmids.

In more specific embodiments, the eukaryotic expression sequence cancomprise a Cytomegalovirus (CMV) promoter. In other aspects, the firstopen reading frame is derived from the genome of a pathogen, including avirus or a bacterium, such as Bacillus anthracis, for example. In otheraspects, the first open reading frame encodes an antigen characteristicof a cancerous cell. In specific aspects, the second antigen can bedisplayed on the surface of the minicell.

Additional embodiments herein include minicells for use in generating animmunogenic response in a subject, comprising a plasmid having an openreading frame, encoding an antigen of interest, and a eukaryoticexpression sequence operably linked to said open reading frame, suchthat said antigen of interest is capable of being expressed in thesubject.

In more specific embodiments, the eukaryotic expression sequencecomprises a Cytomegalovirus (CMV) promoter. In other aspects, the openreading frame is derived from the genome of a pathogen, including avirus or a bacterium, such as Bacillus anthracis, for example. In otheraspects, the open reading frame encodes an antigen characteristic of acancerous cell.

In additional embodiments, the minicells described herein furthercomprise a prokaryotic expression sequence operably linked to said openreading frame, such that said antigen of interest is capable of beingexpressed in the minicell and the subject. In advantageous embodiments,the antigen of interest expressed in the minicell is displayed on thesurface of the minicell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The disclosed invention relates to the use of minicells for thepreparation of immunogenic material. The applications of immunogenicminicells include research, prophylactic, diagnostic and therapeuticapplications. The National Institute for Allergy and Infectious Diseaseshas categorized pathogenic targets for research into three groups,Category A pathogens, Category B pathogens, and Category C pathogens.Any antigen from Categories A, B, and C pathogens can be used with theimmunogenic minicells described herein. As an example, the pathogens ofCategory A, discussed below, provide a number of relevant antigens thatcan be displayed upon the surface of a minicell in order to use theresulting material as an immunogenic composition effective in protectingsubjects from infection of these agents.

In a preferred embodiment, the immunogenic minicells provided hereinencode and are capable of expressing a heterologous antigenic product.Accordingly, in more specific embodiments the described minicells caninclude a vector having a heterologous nucleotide sequence encoding anopen reading frame of an antigen of interest. In even more particularembodiments, the immunogenic minicells described herein include vectorshaving an open reading frame encoding a cancer derived or pathogenderived antigen. As used herein, the term “heterologous” relates to anantigen encoded by the genome of a species other than that from whichthe minicell is derived. Antigens can be encoded by any suitable speciesincluding pathogens, such as viruses, bacteria, fungi, protozoa, and thelike. Antigens can also be encoded by human beings and other mammals.This embodiment is particularly advantageous when it is desirable tohave minicells displaying a cancer antigen.

Immunogenic Minicells

Minicells can be used to immunize subjects. An organism is “immunized”when it is contacted with an immunogen and the organism produces animmune response to the immunogen. The immune response can be protectiveor therapeutic. Examples of protective or therapeutic immune responsesinclude the generation of antibodies, such as neutralizing antibodies,or engendering the proliferation or activity of cytotoxic cells againstthe immunogen.

Immunization strategies can be divided generally into two classes: theuse of whole-killed or attenuated pathogens to present immunogens andthe isolation of immunogens from a pathogen for use as an immunogen.Presentation of whole-killed or attenuated pathogens has many advantagesand frequently produces a robust immune response from an immunizedorganism.

The use of whole-killed or attenuated pathogens as immunogens, however,has certain risks. Perhaps the most serious of these risks involves thepossibility the attenuated immunogen is still sufficiently viable tocause disease in an organism immunized with the attenuated immunogen.Accidental infections with various polio and smallpox vaccines are justtwo examples of this type of risk.

With the advent of molecular biology techniques it became possible toisolate particular antigens from a pathogen for use in an immunogeniccomposition. Often these immunogenic compositions comprise a subunit ofa pathogen that, when presented to an organism, will permit theimmunized organism to generate a protective immune response. Oneextremely successful example of such a subunit immunogen is thehepatitis B subunit vaccine.

Using an immunogenic composition comprising a subunit of a pathogen isadvantageous as it reduces the risk of accidental infection.Unfortunately, isolating one or more subunits from a pathogen for use inan immunogenic composition often leads to a weaker immunogeniccomposition when compared to a whole-killed or attenuated immunogen. Onepossible explanation for this phenomenon holds that the isolated andpreformed antigenic subunit is altered during production and is nolonger in its native confirmation. The resulting immune response to themisshapen immunogen does not serve to prepare a host's immune system toraise a protective immune response against the pathogen of interest.

The use of minicells to present antigens for immunization has severalpotential advantages. For example, immunogenic minicells are able topresent one or more antigenic membrane proteins in their native form toa host's immune system. Native presentation of antigens can be superiorto presenting antigens in a non-native form because the immune systemresponse is more likely to recognize an active pathogen based on theprior exposure to the immunogenic minicells.

In addition to the presentation of antigens in their nativeconformation, immunogenic minicells may present antigens in a mannerthat more accurately mimics antigen presentation by the active pathogenfrom which the antigen of interest was derived. For example, mostnon-enveloped viral pathogens present one or more antigenic epitopes onthe surface of their viral coats in a repeating format. It has beentheorized that mammalian immune systems have adapted to recognize thepresence of repeating antigenic epitopes as the hallmark of an invadingviral pathogen. Immunogenic minicells displaying one or more antigens ontheir surface may be able to more accurately mimic the antigenpresentation of a native virus particle and thus elicit a more robustimmune response than merely providing an antigen to a host in anon-structured format.

Immunogenic minicells have other advantages over standard immunogeniccompositions. Specifically, minicell producing parent cells lines oftencontain immunogenic components, even in the absence of an antigen ofinterest being introduced. For example, the lipopolysaccharide componentof Gram-negative bacteria is known to be extremely immunogenic.Immunogenic minicells displaying an antigen of interest can possiblyelicit a more robust immune response from a host than that elicited by apurified, performed antigen because the host may recognize variouscomponents of the minicell as a foreign antigen. As such, immunogenicminicells present both an antigen of interest and an adjuvant to theimmune system of a host organism. In addition to native minicellcomponents that may act as adjuvants, the immunogenic minicellsdisclosed herein may also be altered to include non-native adjuvantmolecules that further increase the immunogenicity of the minicellcompositions.

In research applications, immunogenic minicells can be used to generateantibodies to an antigen displayed by a minicell. Such antibodies can beused to detect an antigen, which may be a chemical moiety, molecule,virus, organelle, cell, tissue, organ, or organism that one wishes tostudy. Classically, such antibodies have been prepared by immunizing ananimal, often a rat or a rabbit, and collecting antisera therefrom.Molecular biology techniques can be used to prepare antibodies andantibody fragments, as is described elsewhere herein. Single-chainantibody fragments (scFv) may also be identified, purified, andcharacterized using minicells displaying a membrane protein or membranebound chimeric soluble protein.

In prophylactic applications, immunogenic minicells are used tostimulate an immune response from a subject. After administration of theimmunogenic composition disclosed herein, the subject is “pre-immunized”to a pathogen before contact with the pathogen occurs. Thispre-immunization allows the subject to mount a protective immuneresponse to the particular pathogen, thus preventing disease in thesubject.

Certain aspects of the invention involve active immunotherapy. Activeimmunotherapy relies on the in vivo stimulation with an immunogeniccomposition of the endogenous host immune system. Exemplary immunogeniccompositions include immunogens, allergens, toxins, adjuvants, cytokinesand chemokines, all of which allow the host immune system to reactagainst pathogens.

Other therapeutic applications involve passive immunotherapy. Passiveimmunotherapy involves the administration of agents (such as antibodiesor effector cells) directed against an immunogen of a pathogen. Passiveimmunotherapy does not necessarily depend on an intact host immunesystem. Examples of effector cells include T cells; T lymphocytes, suchas CD8+ cytotoxic T lymphocytes and CD4+ T-helper tumor-infiltratinglymphocytes; killer cells, such as Natural Killer (NK) cells andlymphokine-activated killer cells.

Minicell Production

Minicells are anucleated cells that lack chromosomal DNA derived fromthe minicell producing parent cells from which they are produced. Theterm “minicells” encompasses derivatives of eubacterial andarchaebacterial cells that lack parental chromosomal DNA as well asanucleated derivatives of eukaryotic cells. The immunogenic minicellsdescribed herein can be derived from both gram-positive andgram-negative parental cells.

Minicells are produced by minicell producing parent cells. These parentcells undergo cell division in an abnormal manner that produces achromosomal-containing cell and a minicell lacking a copy of theparental chromosome. Minicells are often smaller than their parentcells. For example, minicells produced from E. coli cells are generallyspherical in shape and are about 0.1 to about 0.3 um in diameter,whereas whole E. coli cells are about from about 1 to about 3 um indiameter and from about 2 to about 10 um in length. Table 1 shows avariety of minicell-producing sources useful in the present inventionand discusses the mechanisms by which the minicells are generated.

TABLE 1 Eubacterial Strains, Mutations and Conditions that PromoteMinicell Formation Species Strain Notes References Campylobacter Mayoccur naturally late Brock et al., 1987 jejuni in growth cycle BacillusMutations in divIVB Barak et al., 1999 subtilis locus (inc. minC, minDripX mutations Sciochetti et al., 1999; Lemon et al., 2001 smc mutationsMoriya et al., 1998; Britton et al., 1998 oriC deletions Moriya et al.,1997; Hassan et al., 1997 prfA mutations Pederson and Setlow, 2001Mutations in divIVA Cha et al., 1997 locus B.s. 168 ts initiationmutation Sargent, 1975 TsB143 Bacillus WSBC Induced by exposure to Maieret al., 1999 cereus 10030 long-chain polyphosphate Shigella MC-1 Gemskiet al., 1980 flexneri (2a) S. MC-V Gemski et al., 1980 dysenteriae (1)Lactobacillus Variant minicell- Pidoux et al., 1990 spp. producingstrains isolated from grains Neisseria deletion or overepressionRamirez-Arcos et gonorrhoeae of min homologues al., 2001; Szeto et al.,2001 Escherichia MinA mutations Frazer et al., 1975; coli Cohen et al.1976 MinB mutations and Adler et al., 1967; deletions Davie et al.,1984; Schaumberg et al.; 1983; Jaffe et al., 1988; Akerlund et al., 1992CA8000 cya, crp mutations Kumar et al.; 1979 MukA1 mutation Hiraga etal., 1996 MukE, mukF mutations Yamanaka et al., 1996 hns mutation Kaidowet al., 1995 DS410 Heighway et al., 1989 χ1972, χ 1776 Curtiss, 1980 andχ 2076 P678-54 Temperature-sensitive Adler et al. 1967; cell divisionmutations Allen et al., 1972; Hollenberg et al., 1976 Induced by De Boeret al., 1988 overexpression of minB protein Induced by Pichoff et al.,1995 overexpression of minE protein or derivatives Induced by Ward etal., 1985 overproduction of ftsZ gene Induced by Wang et al., 1991overexpression of sdiA gene Induced by Ramirez-Arcos et overexpressionof min al., 2001; Szeto et genes from Neisseria al., 2001 gonorrhoeaeInduced by exposure Wachi et al., 1999 to EGTA Legionella Induced byexposure Elliot et al., 1985 pneumophila to ampicillin

Minicells are produced by several different eubacterial strains andmechanisms including the overexpression of endogenous or exogenous genesinvolved in cell division, chromosomal replication and partitioning,mutations in such genes, and exposure to various chemical and/orphysical conditions. For example, E. coli cells that overexpress thegene product FtsZ, a protein involved in the regulation of celldivision, form minicells. Minicells are also produced by E. coli cellshaving a mutation in one or more genes of the min locus, which is agroup of genes that encode proteins that are involved in cell division.

Eubacterial cells that have been shown to produce minicells includeEscherichia, Shigella, Bacillus, Lactobacillus, Salmonella, Legionellaand Campylobacter. Bacterial minicell-producing species of particularinterest are E. coli, Salmonella spp., and Bacillus subtilis. Theseorganisms are particularly amenable to manipulation by a variety ofmolecular and genetic methods, with a variety of well-characterizedexpression systems, including many episomal and chromosomal expressionsystems, as well as other factors and elements useful in the presentinvention.

Because minicells lack the chromosomal DNA of the parent cell, RNA andprotein production in the minicells is dependent on the proteinproduction components that segregate into the minicell during formation.It has been alternatively reported that few molecules of endogenous RNApolymerase segregate into minicells and that many RNA polymerasemolecules follow plasmids into minicells. Introduction of an exogenousRNA polymerase to minicell-producing cells enhances expression ofepisomal elements in minicells. Such enhanced expression may allow forthe successful expression of proteins in minicells, wherein suchproteins are expressed poorly or not at all in unmodified minicells. Inorder to maximize the amount of RNA transcription from episomal elementsin minicells, minicell-producing cell lines that express an RNApolymerase specific for certain episomal expression elements may beused. An example of an E. coli strain of this type, designated MC-T7,was created and used as is described in the Examples. Those skilled inthe art will be able to make and use equivalent strains based on thepresent disclosure and their knowledge of the art. Minicell constructionis discussed in more detail in U.S. patent application Ser. No.10/154,951, filed May 24, 2003, which is hereby incorporated byreference in its entirety.

Production of minicells and protein production therefrom may occur usinga variety of approaches or combination thereof. In one approach,minicells are formed and purified. Expression elements contained in theminicells are then stimulated or induced to produce gene productsencoded by the expression elements. In another approach,minicell-producing parent cells containing one or more expressionelements are stimulated or induced to express a protein of interest.Minicell production is subsequently induced in this approach. In yetanother approach, minicell production and protein production areco-induced. The disclosed methods of minicell production teach theexploitation of any timing variable of minicell formation or proteinproduction to optimize minicell and protein production

It is desirable to optimize minicell and protein production fromminicell producing parent cells because these functions can bedetrimental to the host cells. Using inducible minicell and proteinproduction systems permits one to minimize the deleterious effects ofthese procedures. For example, an inducible promoter can be used tocontrol the expression of one or more genes that induce minicellformation. The same inducible promoter or a different inducible promotercan be used to control protein expression from the minicells. Yields ofimmunogenic minicells can be optimized by timing the induction ofminicell production from a minicell producing parent cell line withinduction of protein production from one or more expression vectorsencapsulated within the minicell.

Minicell Purification

A variety of methods are used to separate minicells from parent cells.In general, such methods are physical, biochemical and genetic, and canbe used in combination. The objective of these methods is to minimize oreliminate parental cell contamination in the minicell compositionsproduced using the described methods. For example, minicells areseparated from parent cells using glass-fiber filtration andcentrifugation, both differential and zonal, size-exclusionchromatography, differential sonication, and freeze-thaw cycles.

One centrifugation technique provides for the purification of minicellscan be purified using a double sucrose gradient. The firstcentrifugation involves differential centrifugation, which separatesparent cells from minicells based on differences in size or density. Thepercent of sucrose in the gradient (graduated from about 5 to about20%), Ficol or glycerol is designed to allow only parent cells to passthrough the gradient.

The supernatant, which is enriched for minicells, is then separated fromthe pellet and is spun at a much higher rate (for example, ≧11,000×g).This pellets the minicells and any parent cells that did not pellet outin the first spin. The pellet is then resuspended and layered on asucrose gradient.

The band containing minicells is collected, pelleted by centrifugation,and loaded on another gradient. This procedure is repeated until theminicell preparation is essentially depleted of parent cells, or has aconcentration of parent cells that is low enough so as to not interferewith a chosen minicell application or activity. A variety of buffers andmedia may be used during these purification procedures. These buffersare chosen for their ability to maintain the integrity of the minicellsduring the purification process. Buffers and media used in theseprocedures may serve as an osmo-protectant, stabilizing agent, or energysource, or may contain agents that limit the growth of contaminatingparental cells.

Contaminating parental cells may be eliminated from minicellpreparations by incubation under conditions that selectively killsdividing cells. Because minicells neither grow nor divide, they areresistant to such treatments. An example of conditions that prevent orkill dividing parental cells is treatment of a parent cell culture withan antibacterial agent, such as penicillin. Penicillin prevents cellwall formation and leads to lysis of dividing cells. Other agents may beused to prevent division of parental cells. Such agents include azide.Azide is a reversible inhibitor of electron transport, and thus preventscell division. Additional examples of compounds capable of eliminatingor inhibiting the division of parent cells include D-cycloserine andphage MS2 lysis protein. Khachatourians (U.S. Pat. No. 4,311,797) statesthat it may be desirable to incubate minicell/parent cell mixtures inbrain heart infusion broth at 36° C. to 38° C. prior to the addition ofpenicillin G and further incubations.

Alternatively, lytic phage infection can be used to selectively kill,and preferably lyse, minicell producing parent cells. For example,although minicells can internally retain M13 phage in the plasmid stageof the M13 life cycle, they are refractory to infection and lysis by M13phage. In contrast, minicell producing parent cells are infected andlysed by M13 and are thus are selectively removed from a mixturecomprising parent cells and minicells. For example, a mixture comprisingparent cells and minicells is treated with M13 phage at a multiplicityof infection (M.O.I.) of 5. The infection is allowed to continue to apoint where ≧50% of the parent cells are lysed, preferably ≧75%, morepreferably ≧95% most preferably ≧99%; and ≦25% of the minicells arelysed or killed, preferably ≦15%, most preferably ≦1%.

Another example of a method by which minicell producing parent cells canbe selectively killed, and preferably lysed, exploits the presence of aconditionally lethal gene present in a chromosome of the parent cell.Induction of the chromosomal lethal gene results in the destruction ofparent cells, but does not impact minicells as they lack the chromosomeharboring the conditionally lethal gene. For example, a parent cell maycontain a chromosomal integrated bacteriophage comprising aconditionally lethal gene, such the temperature sensitive repressor genelambda c1857. Induction of this phage, which results in the destructionof the parent cells but not of the achromosomal minicells, is achievedby simply raising the temperature of the growth media. A preferredbacteriophage to be used in this method is one that kills or lyses theparent cells but does not produce infective particles. Expression of atoxic protein or proteins can also be used to selectively kill or lyseminicell producing parental cells. For example, expression of a phageholing gene can be used to lyse parental cells to improve the purity ofminicell preparations.

Modified Forms of Gram-negative Minicells

Gram-negative eubacterial cells and minicells are bounded by an innermembrane (IM), which is surrounded by a cell wall, wherein the cell wallis itself enclosed within an outer membrane (OM). In certainembodiments, it is desirable to use fully intact minicells to stimulatean immunogenic response. In different aspects of the invention, it ispreferred to disrupt or degrade the outer membrane, cell wall or innermembrane of a eubacterial minicell. Such treatments can be used toincrease or decrease the immunogenicity of a minicell.

Eubacterial cells and minicells with altered membranes and/or cell wallsare called “poroplasts” “spheroplasts,” and “protoplasts.” Herein, theterms “spheroplast” and “protoplast” refer to spheroplasts andprotoplasts prepared from minicells. In contrast, “cellularspheroplasts” and “cellular protoplasts” refer to spheroplasts andprotoplasts prepared from cells. Also, as used herein, the term“minicell” encompasses not only minicells per se but also encompassesporoplasts, spheroplasts and protoplasts.

In a poroplast, the eubacterial outer membrane and lipopolysaccharidecomponents have been removed. In a spheroplast, portions of a disruptedeubacterial outer membrane or disrupted cell wall may remain associatedwith the inner membrane of the minicell. The membrane and cell wall ofthe spheroplast is nonetheless porous because the permeability of thedisrupted outer membrane and cell wall has been increased. A membrane is“disrupted” when the membrane's structure has been treated with an agentor incubated under conditions that lead to the partial degradation ofthe membrane, thereby increasing the permeability thereof. In contrast,a membrane that has been “degraded” is essentially, for the applicableintents and purposes, removed. In preferred embodiments, irrespective ofthe condition of the outer membrane and cell wall, the eubacterial innermembrane is not disrupted. Additionally, membrane proteins displayed onthe inner membrane are accessible to compounds that are brought intocontact with the minicell, poroplast, spheroplast, protoplast orcellular poroplast.

Poroplasts

For various applications poroplasted minicells are capable of preservingthe cytoplasmic integrity of the minicell while producing increasedstability over that of naked protoplasts. Maintenance of the cell wallin poroplasted minicells increases the osmotic resistance, mechanicalresistance and storage capacity over protoplasts while permittingpassage of small and medium size proteins and molecules through theporous cell wall.

A poroplast is a Gram-negative bacterium that has its outer membraneremoved. The production of poroplasts involves a modification of theprocedure to make protoplasts to remove the outer membrane. Likeprotoplasts, measuring the total lipopolysaccharide that remains in theporoplast preparation may be used to monitor the removal of the outermembrane. Endotoxin kits and antibodies reactive againstlipopolysaccharide may be used to measure lipopolysaccharide insolution; increasing amounts of soluble lipopolysaccharide indicatesdecreased retention of lipopolysaccharide by protoplasts. This assaythus makes it possible to quantify the percent removal of total outermembrane from the poroplasted minicells.

Several chemical and physical techniques have been employed to removethe outer membrane of Gram-negative bacteria. Chemical techniquesinclude the use of EDTA in Tris to make cells susceptible to hydrophobicagents such as actinomycin. Lactic acid permeabilizes Gram-negativebacteria by disrupting the outer membrane. Physical techniques forremoving the outer membrane include the use of osmodifferentiation tofacilitate the disruption of the outer membrane.

Spheroplasts

A spheroplast is a bacterial minicell that has a disrupted cell wall ora disrupted outer membrane. Unlike eubacterial minicells and poroplaststhat have a cell wall and can thus retain their shape despite changes inosmotic conditions, the absence of an intact cell wall in spheroplastsmeans that these minicells do not have a rigid form.

Protoplasts

A protoplast is a bacterium that has its outer membrane and cell wallremoved. The production of protoplasts typically involves the use oflysozyme and high salt buffers to remove the outer membrane and cellwall. Various commercially available lysozymes can be used in suchprotocols. Measuring the total lipopolysaccharide that remains in theprotoplast preparation is used to monitor the removal of the outermembrane. Commercially available endotoxin kits assays can be used tomeasure lipopolysaccharide in solution; increasing amounts of solublelipopolysaccharide indicates decreased retention of lipopolysaccharideby protoplasts. This assay thus makes it possible to quantify thepercent removal of total outer membrane from the minicells.

For minicell applications that utilize bacterial-derived minicells, itmay be necessary to remove the outer membrane of Gram-negative cellsand/or the cell wall of any bacterial-derived minicell. ForGram-positive bacterial cells, removal of the cell wall may be easilyaccomplished using lysozyme. This enzyme degrades the cell wall allowingeasy removal of now soluble cell wall components from the pelletableprotoplasted minicells. In a more complex system, the cell wall andouter membrane of Gram-negative cells may be removed by combinationtreatment with EDTA and lysozyme using a step-wise approach in thepresence of an osmoprotecting agent. Examples of osmoprotectants includesucrose and glycerol.

It has been found that the concentration of the osmoprotectant sucrose,the cell wall digesting enzyme lysozyme, and chelator EDTA can beoptimized to increase the quality of the protoplasts produced.Separation of either prepared Gram-negative spheroplasts prepared ineither fashion from removed remaining lipopolysaccharide may occurthrough exposure of the spheroplast mixture to an anti-LPS antibody. Theanti-LPS antibody may be covalently or non-covalently attached tomagnetic, agarose, sepharose, sepheracyl, polyacrylamide, and/orsephadex beads. Following incubation, lipopolysaccharide is removed fromthe mixture using a magnet or slow centrifugation resulting in aprotoplast-enriched supernatant.

Monitoring loss of LPS may occur through dot-blot analysis of protoplastmixtures or various commercially available endotoxin kit assays can beused to measure LPS in solution; increasing amounts of soluble LPSindicates decreased retention of LPS by protoplasts. This immunoassaymay comprise a step of comparing the signal to a standard curve in orderto quantify the percent removal of total outer membrane from theminicells. Lipopolysaccharide removal has also been measured by gaschromatography of fatty acid methyl esters.

Minicells from L-form Eubacteria

L-form bacterial strains can be used to prepare antigenic minicells.L-form bacterial strains lack an outer membrane, a cell wall, aperiplasmic space and extracellular proteases. Thus, in L-formEubacteria, the cytoplasmic membrane is the only barrier between thecytoplasm and its surrounding environment.

Segregation of minicells from L-form eubacterial parent cells allows forthe generation of minicells that are at least partially deficient inbarriers that lie outside of the cytoplasmic membrane, thus providingdirect access to components displayed on the minicell membrane. Thus,depending on the strains and methods of preparation used, minicellsprepared from L-form eubacterial parent cells will be similar if notidentical to various forms of poroplasts, spheroplasts and protoplasts.Displayed components that are accessible in L-form minicells include,but are not limited to, lipids, small molecules, proteins, sugars,nucleic acids and/or moieties that are covalently or non-covalentlyassociated with the cytoplasmic membrane or any component thereof.

L-form Eubacteria that can be used in the methods of the inventioninclude species of Escherichia, Streptomyces, Proteus, Bacillus,Clostridium, Pseudomonas, Yersinia, Salmonella, Enterococcus andErwinia.

Assaying Minicells

Levels of minicell production can be evaluated using methods describedherein. Relatively high levels of minicell production are generallypreferred. Minicell production can be assessed by microscopicexamination of late log-phase cultures. The ratio of minicells to normalcells and the frequency of cells actively producing minicells areparameters that increase with increasing minicell production.

Recombinant DNA Expression in Minicells

Recombinant expression of an antigen of interest typically requires theuse of an expression element, such as an expression cassette orconstruct. The expression element contains an open reading frameencoding the antigen of interest that is introduced into an appropriateminicell producing parent cell to generate a minicell expression system.Expression elements of the invention may be introduced into a recipienteubacterial or eukaryotic minicell producing parent cell either as a DNAor RNA molecule, which may be a linear molecule or, more preferably, aclosed covalent circular molecule. Expression from the expressionelement may occur through transient expression of the introducedsequence. Alternatively, permanent expression of the expression elementmay occur through the integration of the introduced expression cassetteinto the chromosome of the minicell producing parent cell.

A variety of recombinant expression systems can be used to produce theantigens for use with the disclosed invention. Any minicell producingparent cell that can be used to express an antigen of interest aresuitable for use with the disclosed methods. Examples of recognizedeubacterial hosts that may be used in the present invention includebacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas,Salmonella, and Serratia.

Eubacterial expression systems utilize plasmid and viral expressionvectors that contain replication sites and control sequences derivedfrom a species compatible with the minicell producing parent cell linemay be used. Suitable phage or bacteriophage vectors include λgt10 andλgt11. Suitable virus vectors may include pMAM-neo and pKRC. Appropriateeubacterial plasmid vectors include those capable of replication in E.coli, such as pBR322, pUC118, pUC119, ColEl, pSC101, and pACYC 184.Bacillus plasmids include pC194, pC221, and pT127. Suitable Streptomycesplasmids include p1J101 and Streptomyces bacteriophages such as C31.Pseudomonas plasmids are also known in the art.

To express an antigen in a eubacterial cell, typically one will operablylink an open reading frame (ORF) encoding an antigen of interest to afunctional promoter. Such promoters can be constitutive or morepreferably, inducible. Examples of constitutive promoters include theint promoter of bacteriophage lambda, the bla promoter of thebeta-lactamase gene sequence of pBR322, and the cat promoter of thechloramphenicol acetyl transferase gene sequence of pPR325. Examples ofinducible eubacterial promoters include the major right and leftpromoters of bacteriophage lambda (P_(L) and P_(R)), the trp, recA,lacZ, lad, and gal promoters of E. coli, the alpha-amylase and thesigma-28-specific promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, and Streptomyces promoters.

Mammalian expression systems utilize host cells such as HeLa cells,cells of fibroblast origin such as VERO or CHO—K1, or cells of lymphoidorigin and their derivatives. Preferred mammalian host cells includeSP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332,which may provide better capacities for correct post-translationalprocessing. Non-limiting examples of mammalian extrachromosomalexpression vectors include pCR3.1 and pcDNA3.1, and derivatives thereofincluding those that are described by and are commercially availablefrom INVITROGEN (Carlsbad, Calif.).

Several expression vectors are available for the expression ofpolypeptides in mammalian minicell producing parent cells. A widevariety of transcriptional and translational regulatory sequences may beemployed, depending upon the nature of the minicell producing parentcell. The transcriptional and translational regulatory signals may bederived from viral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus (CMV), simian virus, or the like, where the regulatorysignals are associated with a particular gene sequence which has a highlevel of expression. Alternatively, promoters from mammalian expressionproducts, such as actin, collagen, myosin, and the like, may beemployed. Transcriptional initiation regulatory signals may be selectedwhich allow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals that aretemperature-sensitive since, by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274,1982; Broach, in: The Molecular Biology of the Yeast Saccharomyces: LifeCycle and Inheritance, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon etal., J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: CellBiology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression,Academic Press, NY, pp. 563-608, 1980).

Expression of polypeptides in eukaryotic hosts generally involves theuse of eukaryotic regulatory regions. Such regions will, in general,include a promoter region sufficient to direct the initiation of RNAsynthesis. Preferred eukaryotic promoters include, the promoter of themouse metallothionein I gene, the TK promoter of Herpes virus, the SV40early promoter, and the yeast gal4 gene sequence promoter.

Expression sequences and elements are also required for efficientexpression. Examples of such sequences include Kozak and IRES elementsin eukaryotes, and Shine-Delgarno sequences in prokaryotes, which directthe initiation of translation (Kozak, Initiation of translation inprokaryotes and eukaryotes. Gene, 1999. 234: 187-208; Martinez-S alas etal., Functional interactions in internal translation initiation directedby viral and cellular IRES elements, Jour. of Gen. Virol. 82:973-984,2001); enhancer sequences; optional sites for repressor and inducers tobind; and recognition sites for enzymes that cleave DNA or RNA in asite-specific manner. Translation of mRNA is generally initiated at thecodon, which encodes the first methionine residue; if so, it ispreferable to ensure that the linkage between a eukaryotic promoter anda pre-selected open reading frame (ORF) does not contain any interveningcodons that encode a methionine. The presence of such codons resultseither in the formation of a fusion protein with an uncharacterizedN-terminal extension (if the AUG codon is in the same reading frame asthe open reading frame) or a frame-shift mutation (if the AUG codon isnot in the same reading frame as the open reading frame).

Expression of Antigenic Proteins

In a preferred embodiment, antigens of interest are expressed andpresented on the surface of minicells. In one embodiment, antigens ofinterest are expressed as integral membrane proteins using a minicellproducing expression system. The expressed antigens are displayed to ahost immune system. Minicell producing cells or minicells harboring anexpression vector are used to express the antigen of interest.

An “expression vector” is typically a nucleic acid encoding an openreading frame operably linked to one or more expression sequences thatdirect the expression of the open reading frame. The term “operablylinked” means that the open reading frame is positioned with respect toexpression sequences so that the amino acid sequence encoded by the openreading frame is faithfully transcribed, producing a gene product. Theterm “gene product” refers to either a nucleic acid (the product oftranscription, reverse transcription, or replication) or a polypeptide(the product of translation) that is produced using the non-vectornucleic acid sequences as a template.

In one embodiment, it is preferable to use an expression construct thatis an episomal expression construct. Minicells produced from a minicellproducing cell line that has been transformed with an episomalexpression construct will contain one or more of the expressionconstructs. These minicells are capable of expressing an open readingframe incorporated into the episomal expression construct. Morespecifically, these minicells will direct the production of thepolypeptide encoded by the open reading frame using the RNA andribosomal machinery that segregated into the minicell at the minicellbudded off from the parent cell. At the same time, any mRNA moleculestranscribed from a chromosomal gene prior to minicell formation thathave been transferred to the minicell are degraded by endogenous RNaseswithout being replaced by new transcription from the (absent) bacterialchromosome.

Chromosomal-encoded mRNAs will not be produced in minicells and will be“diluted” as increasing amounts of mRNAs transcribed from the episomalelement are generated. A similar dilution effect is expected to increasethe relative amount of episomally-generated proteins relative to anychromosome-encoded proteins present in the minicells. It is thuspossible to generate minicells that are enriched for proteins encoded byand expressed from episomal expression constructs.

It is also possible to transform minicells with exogenous DNA after theyhave been prepared or separated from their parent cells. For example,phage RNA is produced in minicells after infection by lambda phage, eventhough replication of lambda phage may not occur in minicells.

Because it is the most characterized minicell-producing species, many ofthese episomal elements have been examined in minicells derived from E.coli. It is understood by practitioners of the art, however, that manyepisomal elements that are expressed in E. coli also function in othereubacterial species, and that episomal expression elements for minicellsystems in other species are available for use in the inventiondisclosed herein.

Eukaryotic and archaebacterial minicells can also be used for expressionof membrane proteins. Use of eukaryotic and archaebacterial minicellsmay be desirable when an antigen of interest expressed in such aminicell has enhanced or altered activity after they undergopost-translational modification processes such as phosphorylation,proteolysis, mystrilation, GPI anchoring and glycosylation.

Expression elements comprising expression sequence operably linked toopen reading frames encoding the membrane proteins of interest aretransformed into eukaryotic cells according to methods and usingexpression vectors known in the art. By way of non-limiting example,primary cultures of rat cardiomyocytes have been used to produceexogenous proteins after transfection of expression elements therefor byelectroporation.

Yeast cells that produce minicells are transformed with expressionelements comprising an open reading frame encoding a membrane proteinoperably linked to yeast expression sequences. Cells that harbor atransferred expression element may be selected using a gene that is partof the expression element that confers resistant to an antibiotic, suchas neomycin.

Alternatively, in one aspect of the invention, bacterial minicells areprepared that contain expression elements that are prepared from shuttlevectors. A “shuttle vector” has sequences required for its replicationand maintenance in cells from two different species of organisms, aswell as expression elements, at least one of which is functional inbacterial cells, and at least one of which is functional in yeast cells.For example, E. coli-yeast shuttle vectors are known in the art andinclude those derived from Yip, Yrp, Ycp and Yep. Preferred E.coli-yeast shuttle vectors are episomal elements that can segregate intoyeast minicells. Particularly preferred are expression vectors of theYep (yeast episomal plasmid) class, and other derivatives of thenaturally occurring yeast plasmid known as the 2 μm circle. The lattervectors have relatively high transformation frequencies and are stablymaintained through mitosis and meiosis in high copy number.

Expression of antigens in eubacterial systems comprising an inner andouter membrane can have the expressed antigenic protein directed toeither the outer membrane, the periplasmic space, the inner membrane, orthe cytoplasm.

Detecting Protein Synthesis in Minicells

Methods for detecting and assaying protein production are known in theart. For example, transformed E. coli minicell-producing cells are grownin LB broth with the appropriate antibiotic overnight. The following daythe overnight cultures are diluted 1:50 in fresh media, and grown at 37°C. to mid-log phase. If it is desired to eliminate whole cells, anantibiotic that kills growing (whole) cells but not quiescent cells(minicells) may be used. For example, in the case of cells that are notampicillin resistant, ampicillin (100 mg per ml is added), andincubation is allowed to continue for about 2 more hours. Cultures arethen centrifuged twice at low speed to pellet most of the large cells.Minicells are pelleted by spinning 10 minutes at 10,000 rpm, and arethen resuspended in M63 minimal media supplemented with 0.5% casaminoacids, and 0.5 mM cAMP, or M9 minimal medium supplemented with 1 mMMgSO₄, 0.1 mM CaCl₂, 0.05% NaCl, 0.2% glucose, and 1 ng per ml thiamine.Labeled (³⁵S) methionine is added to the minicells for about 15 to about90 minutes, and minicells are immediately collected afterwards bycentrifugation for 10 min at 4° C. and 14,000 rpm. Cells are resuspendedin 50 to 100 μg Laemmeli-buffer, and disrupted by boiling and vortexing(2 minutes for each step). Incorporation of ³⁵S-methionine wasdetermined by measuring the amount of radioactivity contained in 1 μl ofthe lysate after precipitation of proteins with trichloroacetic acid(TCA). Minicell lysates (50,000 to 100,000 cpm per lane) are subjectedto 10% PAGE. Gels are fixed and images there of are generated byautoradiography or any other suitable detection systems.

Minicell Modifications

A variety of compounds or moieties can be chemically attached(conjugated) to minicells via membrane proteins that are displayed onthe minicells. The compound to be conjugated to minicells (the“attachable compound”) may of any chemical composition, for example,small molecules, nucleic acids, radioisotopes, lipids or polypeptides.

It is possible to prepare minicells that express transmembrane proteinswith cysteine moieties on extracellular domains. Linkage of the membraneprotein may be achieved through surface cysteinyl groups by, forexample, reduction with cysteinyl residues on other compounds to formdisulfide bridges. If appropriate cysteinyl residues are not present onthe membrane protein they may be introduced by genetic manipulation. Toillustrate, bioactive lysosphingolipids (such as sphingosine,sphingosine-1-phosphate, and sphingosylphosphoryl choline) can becovalently linked to proteins expressed on the surfaces of minicellssuch that these bioactive lipids are on the surface of the minicells.

When the attachable moiety and the membrane protein both have a reducedsulfhydryl group, a homobifunctional cross-linker that containsmaleimide, pyridyl disulfide, or beta-alpha-haloacetyl groups may beused for cross-linking. Examples of such cross-linking reagents includebismaleimidohexane (BMH) or1,4-Di-[3′-(2′-pyridyldithio)propionamido]butane (DPDPB). Alternatively,a heterobifunctional cross-linker that contains a combination ofmaleimide, pyridyl disulfide, or beta-alpha-haloacetyl groups can beused for cross-linking.

Attachable moieties may also be chemically conjugated using primaryamines. In these instances, a homobifunctional cross-linker thatcontains succiminide ester, imidoester, acylazide, or isocyanate groupsmay be used for cross-linking. Examples of such cross-linking reagentsinclude, but are not limited to:Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES);Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSCOCOES);Disuccinimidyl suberate (DSS); Bis-(Sulfosuccinimidyl) Suberate (BS3);Disuccinimidyl glutarate (DSG); Dithiobis(succinimidylpropionate) (DSP);Dithiobois(sulfosuccinimidylpropionate) (DTSSP); Disulfosuccinimidyltartrate (sulfo-DST); Dithio-bis-maleimidoethane (DTME); Disuccinimidyltartrate (DST); Ethylene glycolbis(sulfosuccinimidylsuccinate)(sulfo-EGS); Dimethyl malonimidate.2HCl (DMM); Ethyleneglycolbis(succinimidylsuccinate) (EGS); Dimethyl succinimidate.2HCl(DMSC); Dimethyl adipimidate.2HCl (DMA); Dimethyl pimelimidate.2HCl(DMP); and Dimethyl suberimidate.2.HCl (DMS), and Dimethyl3,3′-dithiobispropionimidate.2HCl (DTBP). Heterobifunctionalcross-linkers that contains a combination of imidoester or succinimideester groups may also be used for cross-linking.

Attachable moieties may also be chemically conjugated using sulfhydryland primary amine groups. In these instances, heterobifunctionalcross-linking reagents are preferable used. Examples of suchcross-linking reagents include, but are not limited to: N-succinimidyl3-(2-pyridyldithio)propionate (DPDP); N-succinimidyl6-[3′-(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP);m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS);succinimidyl 4-[P-maleimidophenyl]butyrate (SMPB); sulfosuccinimidyl4-[p-maleimidophenyl]butyrate (sulfo-SMPB);N-[4-Maleimidobutyryloxy]succinimide ester (GMBS),N-[4-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS);N-[4-maleimidocaproyloxy]succinimide ester (EMCS);N-[4-maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS);N-succinimidyl(4-iodoacetyl)aminobenzoate (STAB);sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SLAB); succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC);succiminidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-caproate)(LC-SMCC); 4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio) toluene(SMPT); and sulfo-LC-SMPT.

As an exemplary protocol, a minicell suspension is made 5 mM EDTA/PBS,and a reducing solution of 2-mercaptoethylamine in 5 mM EDTA/PBS isadded to the minicells. The mixture is incubated for 90 minutes at 37°C. The minicells are washed with EDTA/PBS to remove excess2-mercaptoethylamine. The attachable moiety is dissolved in PBS, pH 7.2.A maleimide crosslinker is added to the solution, which is thenincubated for 1 hour at room temperature. Excess maleimide is removed bycolumn chromatography.

The minicells with reduced sulfhydryl groups are mixed with thederivatized compounds having an attachable moiety. The mixture isallowed to incubate at 4° C. for 2 hours or overnight to allow maximumcoupling. The conjugated minicells are washed to remove unreacted(unattached) compounds having the attachable moiety. Similar protocolsare used for expressed membrane proteins with other reactive groups(e.g., carboxyl, amine) that can be conjugated to an attachable moiety.

Minicell Delivery of DNA Encoding Protective Antigens

Presenting protective antigens to a subject can induce a prophylacticimmune response. The prophylactic immune response is more effective ifthe protective antigens presented are in a native state. The moreaccurately the protective antigen represents the native antigen, themore authentic the immune response generated.

Minicells can be used to deliver plasmid DNA that encodes protectiveantigens from pathogens. Minicells that deliver DNA encoding protectiveantigens produce a more authentic immune response that is more similarto infection-induced immunity. One possible explanation for this resultis that DNA immunization that transfects host cells with a plasmid thatencodes and expresses one or more protective antigens provides antigensto the host's immune system that are in a native state.

Minicell-mediated DNA immunization is advantageous to other methods ofintroducing plasmid DNA to a cell, for example by direct intramuscularor intradermal inoculation, because the plasmid DNA is introduced into agreater number of target cells using minicells rather than directintroduction of the plasmid DNA that encodes protective antigens.

In one embodiment, minicells containing one or more plasmids that encodeone or more antigens of interest are provided to a subject. In onepreferred embodiment, the plasmid encodes one or more heterologousantigens of interest. In another preferred embodiment, the minicellsprepared with the antigen encoding plasmid are designed with one or moreligands on their surface that targets the minicells to a particular celltype. For example, a plasmid carrying minicell may express a cytokine orother ligand specific for a receptor on the target cell. A population ofminicells displaying such a ligand would be targeted to the target celldesignated. Once contacting a target cell, the minicell can then fusewith the target cell and introduce the plasmid DNA contained thereininto the target cell. The control sequences within the plasmid DNA wouldthen direct the synthesis of the antigen of interest from the plasmidDNA.

DNA immunization is superior to immunization by purified antigensbecause it induces a more balanced cellular and humoral immune responsefrom a host. Antigens produced by a host cell are presented in thecontext of class I MHC antigens. This presentation of antigens has beenshown to induce T_(H) cells and CTLs. Accordingly, DNA immunizationshould be more likely to induce a cellular and humoral immune responsefrom an immunized host than merely immunizing with a purified antigenalone. Additionally, foreign antigen introduced into a host by DNAimmunization can be expressed in vivo for several months. Transfectedcells present antigens over a longer period of time than live-attenuatedor nonliving virus vaccines. The increased period of antigenpresentation results in the generation of durable B- and T-cellresponses.

While DNA immunization has its advantages, the technique has certaindisadvantages. For example, the rate at which DNA immunization generatesan immune response, has been reported to be slower than that generatedby the administration of purified antigens. Minicell based DNAimmunization can overcome this deficiency because minicells can bothdeliver the DNA encoding an immunogen and display one or more preformedpurified antigen on the surface of the minicell. While the DNA vectorcan encode a different antigen from that which is displayed on the outersurface of the minicell, in a preferred embodiment, the DNA encodes thesame antigen as is displayed.

One preferred embodiment of the invention relates to minicells thatexpress at least one surface antigen and contain a plasmid designed tofacilitate DNA immunization. Presenting preformed antigens and utilizingDNA immunization can be advantageous over DNA immunization alone becauseit provides an immediate supply of protective antigens to the host'simmune system as well as a long term supply of antigens.

In a preferred embodiment a population of minicells is prepared thatexpresses protective antigens on the membrane of the minicell andcarrying a plasmid within the minicell that encodes one or moreprotective antigens. In preferred embodiments, the plasmid encodes oneor more antigens that are presented on the membrane of the minicell. Astrong promoter encoded by the plasmid, that is active in a variety ofmammalian cell types, typically drives expression of the protectiveantigen. An example of a promoter that can be used with a eukaryoticexpression sequence is a Cytomegalovirus (CMV) promoter. In furtherembodiments, both eukaryotic and prokaryotic expression controlsequences can be present on the plasmid.

Any of the eukaryotic promoters discussed above can be used to prepare aplasmid capable of directing protein expression in a host cells. Plasmidvectors encoding antigens based on the alphavirus replicon system andunder control of the alphavirus subgenomic promoter have been reported.

In certain embodiments, a subject is provided a preparation of minicellsthat present protective antigens on their surfaces. These minicells alsocontain a DNA plasmid that encodes a protective antigen. It is believedthat once these minicells are introduced into the subject, a certainpercentage of the minicells are consumed by macrophages. The proteins ofthe minicell are processed into antigens and displayed to the host'simmune system. The host mounts an antibody response against the antigensof the minicell. A portion of the remaining minicells bind to targetedimmune cells and fuse with those target cells. The plasmid DNA containedwithin these minicells is introduced into the host target cell after thelipid bilayers of the target cell and the minicell fuse. By providingboth preformed antigens and antigens encoded by DNA, this embodiment ofthe invention provides a short-term impetus to generate a humoral immuneresponse and a long term impetus to generate both a humoral and cellularimmune response.

Antigens presented to a host by immunization with DNA can encode anypathogenic open reading frame from any pathogen or source. For example,viral antigens, including antigens that can form virus-like particles,can be used to generate immunogenic compositions with activity agent thepathogen of interest. In addition, DNA encoding bacterial antigens andantigens characteristic of cancerous cells can also be used with theminicells described herein.

Formulation and Administration of Immunogenic Minicells

Formulations of immunogenic minicells include a suitable carrier.Because minicells may be destroyed by digestion or prevented from actingdue to antibody secretion in the gut, they are preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, intradermal, nasal, mucosal,or via suppository. Formulations suitable for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions that maycontain buffers, and solutes which render the formulation isotonic withthe bodily fluid, preferably the blood, of the individual; and aqueousand non-aqueous sterile suspensions which may include suspending agentsor thickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use.

The immunogenic formulations disclosed herein may include adjuvantsystems for enhancing the immunogenicity of the formulation. Adjuvantsare substances that can be used to augment or modulate an immuneresponse. Typically an adjuvant and an antigen of interest are mixedprior to presentation to the immune system. Alternatively, the adjuvantand the antigen are presented separately. Examples of materials suitablefor use in vaccine compositions are provided in Osol, A., ed.,Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton, Pa.(1980), pp. 1324-1341, which reference is entirely incorporated hereinby reference.

Compositions comprising immunogenic minicells are injected into a humanor animal at a dosage of about 0.1-1000 μg per kg body weight. Antibodytiters against antigens of interest are determined by ELISA, using therecombinant protein and horseradish peroxidase-conjugated goatanti-human or animal immunoglobulins or other serologic techniques.Cellular immune responses to immunogenic minicells can also be measuredusing various assays well known to those of ordinary skill in the art.Booster injections are administered as needed to achieve the desiredlevels of protective antibodies or T cells.

Routes and frequency of administration, as well as the dosage ofimmunogenic minicell preparations will vary from individual toindividual. Between 1 and 10 doses may be administered for a 52-weekperiod. Preferably, 6 doses are administered, at intervals of 1 month,and booster administrations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients.

Immunotherapy of hyperproliferative disorders using immunogenic minicellpreparations typically comprises providing a suitable dose of minicellsto a subject in need thereof. Efficacy of such a treatment can bemonitored, as described above, by determining the degree an anti-tumorimmune response results in response to the administration of theimmunogenic minicells. The immune response of an immunized subject canbe monitored by measuring the anti-tumor antibodies in a patient or byimmunogen-dependent generation of cytolytic effector cells capable ofkilling the patient's tumor cells in vitro. Typically, an effective doseof a particular immunogenic minicell composition is capable of causingan immune response that leads to an improved clinical outcome inimmunized subjects as compared to non-immunized subjects.

The immunogenic compositions according to the invention may contain asingle or multiple species of immunogenic minicells where each speciesdisplays a different immunogen. Additionally or alternatively,immunogenic minicells may each display or express one or more immunogen.

Pharmaceutical Compositions

Another aspect of the invention is drawn to compositions, includingpharmaceutical compositions. A “composition” refers to a mixturecomprising at least one carrier, preferably a physiologically acceptablecarrier, and one or more immunogenic minicell compositions. The term“carrier” defines a chemical compound that does not inhibit or preventthe incorporation of the immunologically active peptide(s) into cells ortissues. A carrier typically is an inert substance that allows an activeingredient to be formulated or compounded into a suitable form.Exemplary forms include a pill, a capsule, a gel, a film, a tablet, amicroparticle, a solution, an ointment, a paste, an aerosol, a droplet,a colloid or an emulsion etc.

A “physiologically acceptable carrier” is a carrier suitable for useunder physiological conditions that does not abrogate (reduce, inhibit,or prevent) the immunological activity and properties of the compound.For example, dimethyl sulfoxide (DMSO) is a carrier as it facilitatesthe uptake of many organic compounds into the cells or tissues of anorganism. Preferably, the carrier is a physiologically acceptablecarrier, preferably a pharmaceutically acceptable carrier, in which theimmunogenic minicell composition is disposed.

A “pharmaceutical composition” refers to a composition wherein thecarrier is suitable for use in humans or other animals. The term“pharmaceutically acceptable carrier” includes any medium or materialthat is not biologically or otherwise undesirable. The carrier may beadministered to an organism along with an immunogenic minicellcomposition without causing undesirable effects or interacting in adeleterious manner with the complex or any of its components or theorganism. Examples of pharmaceutically acceptable reagents are providedin The United States Pharmacopeia, The National Formulary, United StatesPharmacopeial Convention, Inc., Rockville, Md. 1990, hereby incorporatedby reference herein into the present application.

The terms “therapeutically effective amount” or “pharmaceuticallyeffective amount” mean an amount sufficient to induce or effectuate ameasurable immunogenic response in the target cell, tissue, or body ofan organism. What constitutes a therapeutically effective amount willdepend on a variety of factors, which the knowledgeable practitionerwill take into account in arriving at the desired dosage regimen.

The compositions of the invention can further comprise other chemicalcomponents, such as diluents and excipients. A “diluent” is a chemicalcompound diluted in a solvent, preferably an aqueous solvent, thatfacilitates dissolution of the composition in the solvent, and it mayalso serve to stabilize the immunogenic composition or one or more ofits components. Salts dissolved in buffered solutions are utilized asdiluents in the art. For example, preferred diluents are bufferedsolutions containing one or more different salts. A preferred bufferedsolution is phosphate buffered saline (particularly in conjunction withcompositions intended for pharmaceutical administration), as it mimicsthe salt conditions of human blood. Since buffer salts can control thepH of a solution at low concentrations, a buffered diluent rarelymodifies the activity of an immunologically active peptide.

An “excipient” is any more or less inert substance that can be added toa composition to confer a suitable property thereto. Suitable excipientsand carriers include, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol cellulose preparations such as,for example, maize starch, wheat starch, rice starch, agar, pectin,xanthan gum, guar gum, locust bean gum, hyaluronic acid, casein potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, polyacrylate, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents can also be included, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. Other suitable excipients and carriers includehydrogels, gellable hydrocolloids, and chitosan. Chitosan microspheresand microcapsules can be used as carriers. See WO 98/52547 (whichdescribes microsphere formulations for targeting compounds to thestomach, the formulations comprising an inner core (optionally includinga gelled hydrocolloid) containing one or more active ingredients, amembrane comprised of a water insoluble polymer, such as ethylcellulose,to control the release rate of the active ingredient(s), and an outerlayer comprised of a bioadhesive cationic polymer, for example, acationic polysaccharide, a cationic protein, and/or a synthetic cationicpolymer; U.S. Pat. No. 4,895,724. Typically, chitosan is cross-linkedusing a suitable agent, for example, glutaraldehyde, glyoxal,epichlorohydrin, and succinaldehyde. Compositions employing chitosan asa carrier can be formulated into a variety of dosage forms, includingpills, tablets, microparticles, and microspheres, including thoseproviding for controlled release of the active ingredient(s). Othersuitable bioadhesive cationic polymers include acidic gelatin,polygalactosamine, polyamino acids such as polylysine, polyhistidine,polyornithine, polyquaternary compounds, prolamine, polyimine,diethylaminoethyldextran (DEAE), DEAE-imine, DEAE-methacrylate,DEAE-acrylamide, DEAE-dextran, DEAE-cellulose, poly-p-aminostyrene,polyoxethane, copolymethacrylates, polyamidoamines, cationic starches,polyvinylpyridine, and polythiodiethylaminomethylethylene.

The immunogenic minicell compositions of the invention can be formulatedin any manner suitable for administration. Immunogenic minicellcompositions may be uniformly (homogeneously) or non-uniformly(heterogeneously) dispersed in the carrier. Suitable formulationsinclude dry and liquid formulations. Dry formulations include freezedried and lyophilized powders, which are particularly well suited foraerosol delivery to the sinuses or lung, or for long term storagefollowed by reconstitution in a suitable diluent prior toadministration. Other preferred dry formulations include those wherein acomposition according to the invention is compressed into tablet or pillform suitable for oral administration or compounded into a sustainedrelease formulation.

When the composition is intended for oral administration but is to bedelivered to epithelium in the intestines, it is preferred that theformulation be encapsulated with an enteric coating to protect theformulation and prevent premature release of the immunogenic minicellcompositions included therein. As those in the art will appreciate, thecompositions of the invention can be placed into any suitable dosageform. Pills and tablets represent some of such dosage forms.

The compositions can also be encapsulated into any suitable capsule orother coating material, for example, by compression, dipping, pancoating, spray drying, etc. Suitable capsules include those made fromgelatin and starch. In turn, such capsules can be coated with one ormore additional materials, for example, and enteric coating, if desired.Liquid formulations include aqueous formulations, gels, and emulsions.

In certain preferred embodiments the immunogenic compositions thatcomprise a bioadhesive, preferably a mucoadhesive, coating. A“bioadhesive coating” is a coating that allows a substance (e.g., aminicell composition) to adhere to a biological surface or substancebetter than occurs absent the coating. A “mucoadhesive coating” is apreferred bioadhesive coating that allows a substance, for example, acomposition according to the invention, to adhere better to mucosaoccurs absent the coating. For example, micronized particles having amean diameter of about 5, 10, 25, 50, or 100 μm can be coated with amucoadhesive. The coated particles can then be assembled into a dosageform suitable for delivery to an organism. Preferably, and dependingupon the location where the cell surface transport moiety to be targetedis expressed, the dosage form is then coated with another coating toprotect the formulation until it reaches the desired location, where themucoadhesive enables the formulation to be retained while thecomposition interacts with the target cell surface transport moiety.

The immunogenic minicell compositions of the invention may beadministered to any organism, preferably an animal, preferably a mammal,bird, fish, insect, or arachnid. Preferred mammals include bovine,canine, equine, feline, ovine, and porcine animals, and non-humanprimates. Humans are particularly preferred. Multiple techniques ofadministering or delivering a compound exist in the art including, butnot limited to, oral, rectal (enema or suppository), aerosol (nasal orpulmonary delivery), parenteral, and topical administration.

Preferably, sufficient quantities of the immunogenic minicellcomposition are delivered to achieve the intended effect. The particularamount of composition to be delivered will depend on many factors,including the effect to be achieved, the type of organism to which thecomposition is delivered, delivery route, dosage regimen, and the age,health, and sex of the organism. As such, the particular dosage of acomposition incorporated into a given formulation is left to theordinarily skilled artisan's discretion.

Those skilled in the art will appreciate that when the immunogenicminicell compositions of the invention are administered as agents toachieve a particular desired immunological result. The desiredimmunological result may include a therapeutic or protective effect.Suitable formulations and methods of administration of therapeuticagents include those for oral, pulmonary, nasal, buccal, ocular, dermal,rectal, or vaginal delivery.

Depending on the mode of delivery employed, the context-dependentfunctional entity can be delivered in a variety of pharmaceuticallyacceptable forms. For example, the context-dependent functional entitycan be delivered in the form of a solid, solution, emulsion, dispersion,micelle, liposome, and the like, incorporated into a pill, capsule,tablet, suppository, aerosol, droplet, or spray. Pills, tablets,suppositories, aerosols, powders, droplets, and sprays may have complex,multilayer structures and have a large range of sizes. Aerosols,powders, droplets, and sprays may range from small (1 micron) to large(200 micron) in size.

Pharmaceutical compositions of the present invention can be used in theform of a solid, a lyophilized powder, a solution, an emulsion, adispersion, a micelle, a liposome, and the like, wherein the resultingcomposition contains one or more of the compounds of the presentinvention, as an active ingredient, in admixture with an organic orinorganic carrier or excipient suitable for enteral or parenteralapplications. The active ingredient may be compounded, for example, withthe usual non-toxic, pharmaceutically acceptable carriers for tablets,pellets, capsules, suppositories, solutions, emulsions, suspensions, andany other form suitable for use. The carriers which can be used includeglucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste,magnesium trisilicate, talc, corn starch, keratin, colloidal silica,potato starch, urea, medium chain length triglycerides, dextrans, andother carriers suitable for use in manufacturing preparations, in solid,semisolid, or liquid form. In addition auxiliary, stabilizing,thickening and coloring agents and perfumes may be used. Examples of astabilizing dry agent includes triulose, preferably at concentrations of0.1% or greater (See, e.g., U.S. Pat. No. 5,314,695, which is herebyincorporated in its entirety). The active compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the process or condition of diseases.

Antigens From Category A Pathogens

Any antigen, including pathogenic and cancer antigens, can be used withthe immunogenic minicells described herein. For example, pathogens fromthe NIAID Category A pathogen list can be used herein, and are discussedin detail below. A brief description of the NIAID Category A pathogen isprovided along with a list of potential antigenic targets and accessionnumbers encoding those targets.

Bacillus anthracis (anthrax)

Anthrax is caused by B. anthracis. This organism is an extracellulartoxinogenic bacterium. An anthrax vaccine comprising minicells thatexpress spore antigens as well as antigens from the anthrax toxins isdisclosed.

Spore Surface

The exosporium is the most external structure of the spore form of B.anthracis. A glycoprotein termed BclA (for Bacillus collagen-like)constituent of the exosporium has been identified. It contains a centralregion presenting similarity to mammalian collagen proteins. BclA is thestructural component of the filaments located at the surface of theexosporium.

Toxins of Bacillus anthracis

Bacillus anthracis secretes two toxins composed of three proteins. Thefirst toxin is the lethal toxin, which is composes of a protectiveantigen (PA) and a lethal factor (LF). The second toxin is the edematoxin, which is composed a PA and an edema factor (EF). The PA(protective antigen) is the common component able to bind and deliver EF(edema factor) and LF (lethal factor) into target eukaryotic cells. EFis a calmodulin-dependent adenylate cyclase and LF is a metalloprotease.

Forms of Bacillus anthracis

The vegetative form of the bacilli is encapsulated. The capsule covers astructural array termed S-layer. The S-layer is composed of two abundantproteins, Sap and EA1. The capsule is composed of at least threeproteins, which genes belong to an operon, are necessary for capsulesynthesis. There is a fourth protein encoded by the same operon thatregulates capsule levels by degrading excess capsule protein.

Provided below is a table of various genes of Bacillus anthracis thatcan be expressed by minicells to generate immunogenic compositions. Notethat this list of genes and their encoded proteins is not intended to bean exclusive list; rather, other genes and proteins from these and otherspecies can be used.

Accession Protein Strain Numbers Bacillus anthracis Bc1A genes ATCC4229AJ516947 CIP5725 AJ516946 RA3 AJ516945 7611 AJ516944 ATCC6602 AJ5169436183 AJ516942 9602 AJ516941 CIPA2 AJ516940 CIP53169 AJ516939 CIP8189AJ516938 CIP7702 AJ516937 Ames AJ516936 PA genes virulence plasmid pX01NC_001496 A2012 plasmid pXO1 NC_003980 BA1024 AF306783 plasmid pX01AF306782 isolate 33 AF306781 isolate BA1035 AF306780 isolate 28 AF306779EF genes virulence plasmid pX01 NC_001496 isolate IT-Carb3-6249 AJ413931isolate IT-Carb1-6225 AJ413930 isolate Sterne M24074 isolate SterneM23179 LF genes virulence plasmid pX01 NC_001496 A2012 plasmid pXO1NC_003980 clone: pLF74 M29081 virulence plasmid pX01 M30210 SAP genesisolate Sterne Z36946 EA1 genes isolate Sterne X99724 Capsule A2012plasmid pXO2, NC_003981 genes CapA, B, & C plasmid pX02 capR AB017611

Clostridium botulinum (Botulism)

Clostridium botulinum is an anaerobic, rod-shaped spore producingbacterium that produces a protein with characteristic neurotoxicity.Antigenic types of C. botulinum are identified by completeneutralization of their toxins by the homologous antitoxin;cross-neutralization by heterologous antitoxins does not occur or isminimal. There are seven recognized antigenic types: A, B, C, D, E, F,and G. Types C and D are not thought to cause human disease, howeverthis has not been definitively established.

Cultures of five of these types apparently produce only one type oftoxin but all are given type designations corresponding to their toxinproduction. Types C and D cross-react with antitoxins to each otherbecause they each produce more than one toxin and have at least onecommon toxin component. Type C produces predominantly C₁ toxin withlesser amounts of D and C₂, or only C₂, and type D producespredominantly type D toxin along with smaller amounts of C₁ and C₂.Mixed toxin production by a single strain of C. botulinum may be morecommon than previously realized. There is a slight reciprocalcross-neutralization with types E and F, and recently a strain of C.botulinum was shown to produce a mixture of predominantly type A toxin,with a small amount of type F.

C. botulinum is widely distributed in soils and in sediments of oceansand lakes. The finding of type E in aquatic environments by manyinvestigators correlates with cases of type E botulism that were tracedto contaminated fish or other seafood. Types A and B are most commonlyencountered in foods subjected to soil contamination. In the UnitedStates, home-canned vegetables are most commonly contaminated with typesA and B, but in Europe, meat products have also been important vehiclesof foodborne illness caused by these types.

Provided below is a table of various genes of C. botulinum that can beexpressed by minicells to generate immunogenic compositions.

Accession Protein Strain Numbers Clostridium Neurotoxin 5′ end M27892botulinum Type A isolate Kumgo, light chain- AY166872 partial cdsSynthetic construct AF464912 Neurotoxin Isolate 1436 AF295926 Type Bisolate 13280 AF300469 isolate 667 AF300468 isolate 519 AF300467 isolate593 AF300466 isolate 588 AF300465 Neurotoxin Isolate 6813 D49440 Type CBotulinum bacteriophage X62389 Bacteriophage c-st D90210 C2 toxincomponent-I and D88982 component-II C2 toxin (component-I) D63903Neurotoxin BVD/-3 X54254 Type D Neurotoxin Isolate 35396 AB082519 Type EHazen 36208 X70815 VH Dolman X70818 NCTC 11219 X62683 Beluga X62089Neurotoxin 202F Y10770 Type F proteolytic F Langeland X70821non-proteolytic Hobbs FT10 X70820 non-proteolytic Craig 610 X70816 202FM92906 Neurotoxin synthetic sequence based on AX608812 Type G Wild type113/30, NCFB 3012 X74162

Yersinia pestis

Yersinia pestis is the causative agent of plague. The nucleotidesequence of the organism's genome has been elucidated. “Genome sequenceof Yersinia pestis, the causative agent of plague,” Nature 413 523-527.

Y. pestis has been extensively studied and this work provides a numberof potential targets for a subunit vaccine. Prime candidates include theF1 antigen (cfa1), pla, the V antigen (LcrV), and Yops. The YscC proteinhas been advanced identified as residing in the outer member of theorganism and as such could also provide a potential vaccine target. SeeClin Microbiol Rev. 10(1):35-66 (1997) for a more complete review.

Provided below is a table of various genes of Y. pestis that can beexpressed by minicells to generate immunogenic compositions.

Accession Protein Strain Numbers Yersinia Complete Genome CO92 NC_003143pestis KIM NC_004088 F1 Antigen (caf1) caf1, caf1M, caf1A and X61996caf1R pla gene KIM AF053945 V antigen CO-92 Biovar Orientalis NC_003131Angola AF167310 Pestoides AF167309 Yops CO-92 Biovar OrientalisNC_003131 KIM5 AF074612 KIM AF053946

Variola Major (Smallpox) and Other Pox Viruses

The smallpox virus genome has been completely sequenced.

Accession Organism Protein Strain Numbers Variola major GenomeIndia-1967, ssp. major NC_001611 (smallpox) sequence strainBangladesh-1975 L22579 Vaccinia Virus Genome Ankara U94848 SequencesOther genomic DNA, 42 kbp D11079 Sequences Various genes D00382 Variousgenes M36339 Various genes AF411106 Various genes AF411105 Various genesAF411104 a13L ortholog gene for p8 AJ309902 AJ315004 A36R gene forp43-50 AF120160 protein DNA glycosylase (D4R and L24385 D5R) genesVarious genes M57977 A33R (A33R) gene AF226618 L1R (L1R) gene AF226617serine proteinase inhibitor D00582Francisella tularensis (tularemia)

Francisella tularensis is a small gram-negative coccobacillus. There aretwo main serotypes: Jellison Types A and B. Type A is considered themore virulent form. F. tularensis may be aerosolized in dry or wet form.

Viral Hemorrhagic Fevers

Arenaviruses

Arenavirues are enveloped. The surface of the virion envelope is studdedwith glycoprotein projections that consist of tetrameric complexes ofthe viral glycoproteins GP1 and GP2. Obtaining gene and amino acidsequences for these proteins from each of the arenaviruses listed belowwould be helpful in supporting a vaccine patent application.

LCM

Lymphocytic chorio meningitis (LCM) is caused by the lymphocytic choriomeningitis virus (LCMV). Studies in mice have shown that passiveimmunity is effective in protecting suckling mice from LCMV challenge.Because a protective immune response has been demonstrated, it may bepossible to use minicell technology to produce large amounts ofantibodies to treat victims of LCMV. Of course, a vaccine effectiveagainst LCMV would be a primary goal. LCMV appears to interact with CD+8cells as part of its life cycle. The viral protein or proteins involvedin this interaction would make prime targets for vaccine antigens usingthe disclosed minicell technology.

Organism Protein Strain Accession Numbers LCMV GP1 WE AJ233161 DocileAJ249159 Docile AJ249158 GP-C CHV2 U10158 CHV3 U10159 CHV1 U10157Junin Virus

The Junin virus is a member of Arenaviridae. It is pleomorphic,enveloped globular virions 110-130 nm in diameter, linear,single-stranded, two-segmented RNA. Junin virus found mainly inArgentina and causes Argentinean hemorrhagic fever. Potential antigenictargets from the Junin virus include the GP1 and GP2 envelopeglycoproteins. The NP, L and Z proteins are thought to be internal tothe viral particle and would seem to be less likely targets.Nevertheless, these proteins can also be used to generate immunogeniccompositions.

Organism Protein Strain Accession Numbers Junin Virus G1 (Partial)PH3190 AF264235 PH7994 AF264234 PAn14823 AF264233 PH2412 AF264232 GP-CMC2 D10072 NP Vaccine Strain U70804 Segment L N/A N/A Z N/A N/A

Other arenavirues of interest include the Machupo virus, the Guanaritovirus and the Lassa Fever genomic information and accession numbers forantigens of interest are provided below.

Accession Organism Protein Strain Numbers Machupo Viruso S SegmentCarvallo AY129248 (Glycoprotein) Carvallo AF485260 nucleocapsid proteinAA288-77 X62616 Gaunarito S Segment INH-95551 AY129247 (Glycoprotein)INH-95551 AF485258 nucleocapsid protein VHF-5603 AF204207 (Partial)VHF-1150 AF204206 S-16995 AF204205 VHF-3990 AF204204 Lassa Virus GP1803213 AF181854 LP AF181853 11620 M15076 Segment L Josiah NC_004297Segment S Josiah NC_004296 GP-C AV AF246121 Nucleoprotein 11620 J04324803213 AF181854 LP AF181853Bunyaviruses:

Bunyaviruses are spherical particles that display surface glycoproteinprojections of 5 to 10 nm, which are embedded in a lipid bilayeredenvelope approximately 5 to 7 nm thick. Depending on the virus, therecan be from 270 to 1,400 glycoprotein spikes per virion. The spikes aregenerally thought to consist of heterodimers of the viral glycoproteinsG1 and G2. The G1 and G2 proteins are decorated with N-linkedcarbohydrates, so this may make these viruses less attractive astargets. Two bunyaviruses of interest are the hantavirus and the RiftValley fever virus. Sequence information for these viruses is providedbelow.

Accession Organism Protein Strain Numbers Hantavirus G1 & G2 cl-1 D2552984FLi AF366569 B-1 X53861 M Polypeptide 84FLi AF345636 MF-43 AJ01164884FLi AF366569 Ls136V AJ011647 Rift Valley Fever Complete genomeZH-548M12 NC_002044 Virus G1 & G2 Rift Valley Fever M11157 VirusFlaviviruses

There are a number of members of flavividae that are particularlyattractive to serve as targets for a minicell immunogenic composition.Examples of pathogenic flaviviruses include the hepatitis C virus, St.Louis encephalitis virus, dengue, and a number of other encephalitisviruses. Sequence information for these viruses is provided below.

Accession Organism Protein Strain Numbers Hepatitis C Complete genomeM1LE AB080299 H77 NC_004102 E protein JB2-8 AJ511254 JB28-1 AJ511253JB15-10 AJ511252 M protein N/A N/A C protein Gabonese S73403 GaboneseS73404 Gabonese S73421 St Louis encephalitis E protein (partial) BFS508AF112392 FL79-411 AF112391 Hubbard AF112389 M protein N/A N/A C proteinN/A N/A Dengue Complete genome 814669 AF326573 rDEN4del30 AF3268272Adel30 AF326826 N/A NC_001474Filoviruses

Filoviruses are enveloped viruses that can cause hemorrhagic fevers.Exemplary filoviruses include the Ebola and Marburg viruses. There arefour species of Ebola-like viruses: Zaire, Sudan, Reston, and Côted'Ivoire. There is only one representative of the Marburg filovirus.Each of these viruses contains VP40, GP, and VP24 proteins that arethought to be membrane-associated proteins. These proteins arecandidates for vaccine targets using the disclosed minicell technology.

Accession Organism Protein Strain Numbers Zaire Ebola virus Completegenome Mayinga NC_002549 Mayinga AY142960 Sudan Ebola virus NP BonifaceAF173836 SP Boniface U28134 L Maleo 1979 U23458 GP Maleo U23069 RestonEbola virus Complete genome Pennsylvania NC_004161 Reston AB050936 Coted'Ivoire Ebola Complete genome N/A N/A virus Marburg virus Completegenome Popp NC_001608 NP, VP35, VP40, GP, Popp Z29337 VP30, VP24, Lgenes Musoke Z12132

EXAMPLES Example 1 Construction of an Inner Membrane Expression VectorpMPX200

The pMPX-200 expression vector was designed to express a gene product ofinterest as an inner membrane-bound, periplasmic exposed fusion protein.The nucleotide sequence of this vector is provided as SEQ ID NO: 1 andis shown without a coding sequence of interest. To construct anexpression vector containing a coding sequence of interest, one insertsthe gene or coding sequence of interest into the SalI/XbaI restrictionregion of the pMPX200 expression vector to create a chimeric fusion withthe transmembrane domain (TMD) of toxR.

Example 2

Construction of an Outer Membrane Expression Vector pMPX201

The pMPX-201 expression vector was designed to express a gene product ofinterest as an outer membrane-bound, extracellular exposed fusionprotein. The nucleotide sequence of this vector is provided as SEQ IDNO: 2 and is shown without a coding sequence of interest. To constructan expression vector containing a coding sequence of interest, oneinserts the gene or coding sequence of interest into the XhoI/XbaIrestriction region of the pMPX201 expression vector to create a chimericinsertion into lamB.

Example 3 ToxR-Bacillus anthracis PA Fusion Protein

Immunogenic minicells expressing the soluble Bacillus anthracisprotective antigen (PA) on the outer membrane of an E. coli derivedminicell are prepared and purified according to the protocols discussedin Examples 1 and 3. E. coli derived minicells transformed with theexpression vector discussed in Example 1 but lacking the ToxR::PA fusionprotein coding sequence are also prepared as a negative control. The twospecies of minicells are then formulated for intramuscular injection.

Minicells displaying the ToxR::PA fusion protein are provided to a groupof test subjects. On day 1 the test subjects are provided with aninitial dose of an inoculum comprising the ToxR::PA fusion proteinexpressing minicells. Control subjects are provided with an initial doseof an inoculum comprising minicells that do not express the ToxR::PAfusion protein. A booster is provided to each group of animalsapproximately two weeks later. Approximately 14 days after the initialimmunization, blood is taken from the test and control subjects and usedin an ELISA to determine if the subjects have mounted an antibodyresponse against the inoculum. Serum analyzed by ELISA indicates thatthe test subjects mount an antibody response against the fusion proteininoculum. Serum taken from the control animals indicates that while theanimals mounted an antibody response against the minicells themselves,they have not produced antibodies that react with the ToxR::PA fusionprotein.

Lymphocytes isolated from the test groups of animals are shown to bereactive with the fusion protein expressing minicells. Lymphocytesisolated from the control group animals are shown not to be reactivewith the fusion protein expressing minicells.

Approximately three weeks after the initial inoculation, the testanimals are challenged with live Bacillus anthracis. The test subjectsare completely protected from the development of anthrax symptoms whilethe control subjects die from anthrax.

Example 4 Minicell-Based DNA Delivery of GFP-Encoding Plasmids in aMouse

Introduction

The purpose of this study was to demonstrate the capability of bacterialminicells to deliver plasmid DNA to cells in vivo for expression of atarget protein. For the purpose of these experiments, nucleic acidencoding Green Fluorescent Protein (GFP) was used as the target protein.The read out for these experiments was the production of antibodiesagainst GFP. The theory of the experiment is that if GFP were expressedby cells in a live mouse, the mouse's immune system would mount animmune response against the produced GFP, resulting in an antibodyresponse. Without successful delivery of the plasmid DNA by the minicelland the subsequent expression by the mouse cell, an antibody responsewould not be obtained.

Methods

Three general types of minicells were created. Each of the three typesof minicells contained a plasmid having nucleic acid encoding GreenFluorescent Protein (GFP). The first type of minicell included a plasmidcontaining a eubacterial expression sequence operably linked to the GFPnucleic acid, and did not contain a eukaryotic expression sequence. Thisplasmid was therefore designed to produce GFP protein in the minicelland not the animal target (administered to Group No. 4). The second typeof minicell included a plasmid containing a eukaryotic expressionsequence (CMV promoter) operably linked to the GFP nucleic acid, asopposed to a eubacterial expression sequence. This expression cassettewas designed to drive the in vivo expression of the GFP protein in thecells within the target mouse (administered to Group No. 5). The thirdtype of minicell included a plasmid having both eubacterial andeukaryotic expression sequences operably linked to the GFP nucleic acid.This third expression cassette was designed to drive expression of GFPin the minicell and the in vivo expression of GFP in the cells withinthe target mouse (administered to Group No. 6).

The study involved the intramuscular (IM) injection of mice on day onefollowed by booster injections on day 14 and 28. The mice were thentaken down on day 35 and serum was collected for determination ofantibody titer. Six groups of three mice were used for this experimentand the experiment was carried out twice for a total of six mice pergroup. The first group was a naïve control meaning that they did notreceive any injections. The second group was injected with controlminicells that did not have any GFP protein or GFP plasmid DNA. Thethird group was injected with the naked GFP plasmid alone (eukaryoticexpression sequence operably linked to GFP nucleic acid). This groupserved as the positive control group as it is well known that IMinjections of naked DNA are capable of inducing an antibody responseagainst proteins encoded by the delivered plasmid. The fourth group wasinjected with minicells containing the expressed GFP protein, todetermine the ability of minicells to deliver protein. The fifth groupwas injected with minicells containing the plasmid having a eukaryoticexpression sequence operably linked to GFP nucleic acid. The final groupwas injected with minicells containing both the plasmid having aeukaryotic expression sequence operably linked to GFP nucleic acid, aswell as the expressed GFP protein to determine if delivery of GFPprotein in combination with the GFP plasmid would enhance the response.The groups are outlined in the table below. All groups were normalizedto contain equal amounts of plasmid and protein.

GROUPS OF MICE Group # Injection 1 Naïve mice 2 Empty minicell control 3Naked GFP plasmid DNA 4 Minicell containing GFP protein 5 Minicellcontaining GFP plasmid 6 Minicell containing both GFP protein and GFPPlasmid

Following the take down of the mice, serum was collected from all miceand prepared for antibody titer analysis. Antibody titer analysis wascarried out using standard ELISA techniques. Purified GFP protein wasused in the ELISA as the substrate for the detection of mouse GFPantibodies and HRP conjugated anti-mouse antibodies were used to detectthe anti-GFP antibodies produced by the mice. Read out was measuredusing an ELISA plate reader and the data was converted to titers usingstandard equations.

Results

As expected, the serum from groups one and two did not contain anydetectable antibodies against the GFP protein. Groups three, four, andfive all obtained a measurable titer and were statistically the same.All three groups were able to induce an antibody response, whichindicates that the naked plasmid DNA as well as the plasmid DNA in theminicell was delivered to an in vivo target and GFP was produced,resulting in an antibody response. Surprisingly, the level of responseto the minicell with a combination of both GFP protein and GFP plasmid(group 6) was much higher than all other groups. The titer was 10× theresponse of groups 3, 4, and 5. These data definitively demonstrate theability of minicells to deliver plasmid DNA to an in vivo target andprotein production from the plasmid upon delivery in the target cell.

What is claimed is:
 1. A fully intact eubacterial minicell for use ingenerating an immunogenic response in an animal, comprising a plasmidhaving an open reading frame encoding a viral antigen of interest, and aeukaryotic expression sequence operably linked to said open readingframe, such that said antigen of interest is expressed in said animal.2. The minicell of claim 1, wherein said eukaryotic expression sequencecomprises a CMV promoter.
 3. The minicell of claim 1, wherein saideukaryotic expression sequence comprises a stress inducible an induciblepromoter.
 4. The minicell of claim 1, wherein said open reading frame isfrom the genome of a virus.
 5. The minicell of claim 4, where said virusis selected from the group consisting of HIV, smallpox virus, HepatitisA, Hepatitis B, Hepatitis C, influenza virus, varicella zoster virus,Herpes Simplex Virus, adenovirus, rhinovirus, rinderpest, exhovirus,rotavirus, respiratory syncitial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus,mumps virus, measles virus, polio virus, and rubella virus.
 6. Theminicell of claim 1, further comprising a prokaryotic expressionsequence operably linked to said open reading frame, such that saidantigen of interest is expressed in the minicell and the animal.
 7. Theminicell of claim 6, wherein said antigen of interest expressed in theminicell is displayed on the surface of the minicell.